BIOCHEM Lab Final- Lab 9, prelab 6 , prelab 7, Prelab 8, prelab10

How many grams of KCl do you need to make 450ml of a 0.5M Tris, 50mM KCl 10x stock solution? (MW tris = 121.1g/mole, MW KCl =74.6g/mole) [round to the nearest tenths place, record your answer as a number (gram units are supplies in the question)]

+/-0(1.7-1.7) answer: 1.7

Aλ = 9

-log10(I/I0) = bcE

Pure protein has a ratio of 9

0.6. Too HIGH (Above 0.6) An ideal 260/280 ratio for common proteins is 0.6. Higher ratios may indicate the contamination of isolated proteins with DNA Too LOW (Below 0.6): Nucleic acids have a higher absorbance at 260 nm than proteins, so a low A260/A280 ratio suggests that the sample contains more nucleic acids than proteins.

Given that a 1mg/ml pure sample of mouse IgG has an absorbance at A280 of 1.34, what is the concentration (in mg/ml) of an unknown sample of mouse IgG if you measured an A280 of 0.88? 9

0.66 1/1.34 = X/0.88 X=0.656

What are the values of Aλ=bcE 9

Aλ= absorbance at a particular wavelength b= path length of cuvette (1cm) c= concentration of sample Eλ= molar extinction at a particular wavelength

A mixture of DNA and protein gives an A260 of 1.13 and an A280 of 1.98. What is the concentration of the protein? 9

1.55*A280 - 0.76*A260 1.55*1.98 - 0.76*1.13 = 2.21 mg/ml

Protein mg/ml = 9

1.55*A280nm - 0.76*A260nm

Looking at the "Reaction Rate as a Function of Enzyme Concentration" section in the lab protocol, which assay would you predict to have the highest LDH activity and why? 10

Assay 1, it has more LDH present in the test tube

Looking at the "Reaction Rate as a Function of Substrate Concentration" section in the lab protocol, which assay would you predict to have the highest LDH activity and why?

Assay 6, because it has more lactate in the test tube

Pure DNA has a ratio of 9

1.7 to 2.0. Too Low (Below 1.7): A 260/280 ratio below 1.7 indicates that the DNA sample may be contaminated with proteins, which absorb more strongly at 280 nm. This could be due to incomplete removal of proteins during the DNA purification process, or the presence of proteins that co-precipitated with the DNA. In this case, it is recommended to repeat the purification process, making sure to remove any protein contaminants. Too High (Above 2.0): A 260/280 ratio above 2.0 indicates that the DNA sample may be very pure, with little or no protein contamination. However, it could also indicate the presence of RNA, which absorbs at 260 nm but not at 280 nm. RNA contamination can occur during the DNA isolation process, especially if the sample contains high levels of RNA, or if the DNA isolation protocol is not optimized to remove RNA. In this case, it may be necessary to perform an additional step, such as DNase treatment, to remove any remaining RNA.

To make 2 ml of a 1:100 dilution of a stock solution in buffer you would combine

2 ml * 1/100 = 0.02 ml = 20 ul stock 2 ml total volume - 0.02 ml stock = 1.98 ml buffer

Shown below is a schematic representation of a SDS gel. Thickness of bands correlate with amounts of protein in the band. What is the purify of band B in the E2 sample? 6

20% **** 3B + C = 100 % A+B+C = 100%, B=C, A=3B (In the E2 column/well) B = 1/5 (20%), A = 3/5, C = 1/5 (20%) Replace all occurrences of B in A+B+C=100% =. A+C+C=100% B=C A=3B Simplify A+C+C=100% A+2C=100% B=C A=3B A+2C=1 B=C A=3B Replace all occurrences of B in A=3B with C A=3(C) A+2C=1 B=C Multiply 3 by C A=3C A+2C=1 B=C Replace all occurrences of A with 3C in each equation. Replace all occurrences of A in A+2C=1 with 3C (3C)+2C=1 A=3C B=C 5C=1 A=3C B=C Divide each term in 5C=1 by 5 and simplify. C=1/5 A=3C B=C Replace all occurrences of C with 1/5 in each equation. B=1/5 A=3/5 C=1/5 Solve the above equations, & you get the answer

Monitor DNase 1 activity at 9

260nm

Using the stock solution from the previous question, what is the mM concentration of the KCl in the working solution? [record your answer as a number only (mM units are supplied in the question)]

5

What is the estimated molecular weight of a protein in a band located 4.6 cm from the top of the gel? 6

50kD Draw a line up from the distance (cm specified in the question) on the x-axis until it intersects the standard curve. Then interpolate across from the point of intersection to the y-axis to find the corresponding molecular weight. ********************** If the curve is nearly linear, it can be described by the formula y = mx + b, where y is the log MW, m is the slope, x is the Rf, and b is the y-intercept, Currently, the supporting medium of choice for almost all protein electrophoresis applications (and many nucleic acid electrophoresis applications) is polymerized acrylamide.

Spectrophotometer measures cuvette @ _____ o'clock position of rotating turret 9

9 o'clock

A student used western blotting to confirm the presence of C. elegans SMAP5 in samples run on SDS-PAGE & transfered to a nitrocellulose membrane. The image above shows all interactions prior to the addition of substrate. Which component of the image represents SMAP5? 7

A student used western blotting to confirm the presence of C. elegans SMAP5 (antigen) in samples run on SDS-PAGE Black circle Response Feedback: In this experiment, SMAP5 is the antigen so it is represented by the black circle.

Which of the following bind proteins nonspecifically? Choose ALL that apply 7

Binds proteins nonspecifically: Coomassie blue Nitrocellulose membrane Ponceau S PVDF membrane

Calculating A260/A280 ratio is a good indicator of 9

Calculating A260/A280 ratio is a good indicator of purity. The A 260/280 ratio is often used as a measure of the purity of DNA samples since it reflects the ratio of absorbance at 260 nm and 280 nm, which are wavelengths where DNA and protein absorb light, respectively.

Molecular weights of the 6 bands in the SDS gel below are 6kD, 14kD, 31kD, 45kD, 66kD, & 97kD. Which protein has the MW of 31kD? 6

Carbonic anhydrase has the approximate MW of 31 kD. Molecular weights of the 6 bands in the SDS gel below are: Bottom to top: Lysozyme 6 kD, Soybean trypsin inhibitor 14 kD, Carbonic anhydrase 31 kD, Ovalbumin 45 kD, Bovine serum albumin 66 kD, Phosphorylase b 97 kD.

dsDNA => DNase 1 ==mg2+==> 9

Cleave backbone, double stand break Increase A260 reading

A student wants to use western blotting to confirm the presence of chicken collagen type II in samples run on SDS-PAGE & transfered to a nitrocellulose membrane. If they use Mouse anti-collagen type II MAb & TMB as part of a successful western blot, which of the following could the "Green Y with purple knob" in the figure be? 7

Donkey anti-mouse IgG-HRP Mouse anti-collagen type II MAb is the primary antibody, so an appropriate 2ndary antibody (green Y) will need to be anti-mouse IgG. TMB is the substrate for colorimetric detection, so the enzyme (purple knobs) conjugated to the 2ndary antibody will need to be HRP. Of the available options, Donkey anti-mouse IgG-HRP is the only enzyme-conjugated 2ndary antibody (green Y with purple knobs) that meets the requirements.

Which of the following statements is correct about the EXPECTED result of Ponceau S staining after the transfer? 7

Expect to see more bands in the lanes containing the wash fraction samples than those containing the elution fraction samples. Many bands in the lane containing your GCE sample. See the molecular weight ladder bands.

Based on the data above, a resolving gel higher than 12.5% is needed to resolve 20 kD & 30 kD proteins. 6

FALSE Per the graph, the 30 kD & 20 kD proteins would have migrated to approximately 6.8 cm & 8.6 cm respectively. 1.8 cm is plenty of space to see the separation of the bands. *************************

An elution fraction from a Ni+2 agarose column that has a high rGFP florescence will also have a high purity.

False. activity depends on the amount of active rGFP present in the fraction purity of rGFP depends upon the absence of contaminants

A highly positive charged protein will bind a anion exchanger and elute of with a high salt buffer.

False. **

You measure the absorbance of 1 ml of the 1:100 dilution at the appropriate wavelength. The spectrophotometer reading is 0.400. If you know that a 1 mg/ml solution gives a reading of 1.60 at that wavelength, what is the concentration of your stock solution (in mg/ml)? Hint #1: You don't need to use Beer-Lambert equation to solve this. (See lecture 1 notes if confused.) Hint #2: Don't forget about the dilution.

Feedback: 1) Use a ratio to solve for the concentration of the diluted solution: (1 mg/ml)/1.6 = (x mg/ml)/0.400 2) Multiply the concentration the diluted solution by 100 (because it was a 1:100 dilution) to get the concentration of the stock solution.

Which column would you use to purify a 175kd negatively charged tagged protein from a 220kd negatively charged protein?

Gel filtration columns separate based upon size. The stated difference between the two proteins is 175kD and 220 kD, the G200 matrix will allow the 175 kD to enter the beads and exclude the 220 kD Ni+2 columns are used to separate His6 tagged proteins - the question does not specify the type of tag on the protein (there are other tags like a myc tag or a FLAG tag. Ion exchange columns separate based upon charge. Both proteins in this question are negatively charged.

In the presence of Mg2+, DNase 1 produces single strand breaks in the DNA backbone. What happens to the A260 reading of a DNA sample? 9

In increases...** In the presence of Mg2+, DNase I produces single-strand breaks in the DNA backbone, leading to the fragmentation of the DNA. This fragmentation can affect the A260 reading of a DNA sample, which is a measure of the amount of nucleic acid present in a solution. As the DNA becomes fragmented, the # of intact DNA decreases, which can lead to a decrease in the A260 reading. This decrease in the A260 reading may not be proportional to the degree of DNA fragmentation, however, since other factors such as impurities or incomplete digestion may also affect the A260 reading. Therefore, it is important to use other methods, such as gel electrophoresis or fluorometry, to confirm the degree of DNA fragmentation in a sample.

The expression & purification of rGFP started with the growth of the bacterial culture & ended with the elution of rGFP off the Ni+2-agarose column. Assuming the column was not overloaded, which of the following changes to the procedure would result in an increase in the yield of rGFP in the elution fractions? Select all that apply. 6

Induce the culture longer than 3 hours before harvesting. Use a 30ml pellet instead of a 15ml pellet. Increasing the induction period beyond 3 hours (more rGFP produced) or using a pellet larger than 15ml (more bacteria w/ rGFP present) would result in more rGFP being put into the procedure which should result in a higher ending yield. Because you didn't overload the column, Increasing the Ni+2 agarose bed volume will NOT increase the yield of rGFP purified. Wash buffer removes unbound proteins from the Ni+2 column. It DOES NOT increase the overall yield of rGFP bound or eluted.

Which column would you use to purify a 32kd (+) charged tagged protein from a 35kd (-) charged protein?

Ion exchange columns separate based upon charge. Ni+2 columns are used to separate His6 tagged proteins - the question does not specify the type of tag on the protein (there are other tags like a myc tag or a FLAG tag. Gel filtration columns separate based upon size, not charge. The difference in the stated protein sizes is not enough to be resolved with a typical gel filtration column.

lactate dehydrogenase 9

Lactate + NAD+ = Pyruvate + NADH NADH absorbs at 340nm NAD+ does not absorb at 340nm ************ The lactate detection by LDH catalysis is based on the NADH quantification. Since molecule can be detected both electrochemically and optically, we used UV-Visible spectrometry as control technique for NADH quantification by measuring the solutions absorbance at 340 nm. What is specific activity of LDH? LDH activity is reported as nmole/min/mL = milliunit/mL One unit of LDH activity is defined as the amount of enzyme that catalyzes the conversion of lactate into pyruvate to generate 1.0 μmole of NADH per minute at 37 °C.

Larger proteins elute off a gel filtration column in earlier fractions.

Larger proteins elute off a GEL FILTRATION column in early fractions because they are excluded from the pores in the beads. TRUE

Larger proteins elute off a Ni+2 agarose column in early fractions.

Larger proteins elute off a GEL FILTRATION column in early fractions. FALSE

Microplate reader measures _____ at ____wavelength 9

Measure multiple samples (wells) at 1 wavelength

Single cell spectrometer measures ______ at _____ wavelengths 9

Measures one sample (cuvette) at multiple wavelength light causes no damage

Monitor bacterial growth by 9

Monitor bacterial growth by: turbidity dominates the readings, not absorbance **************

What are the typical uses of UV- visible spectroscopy? 9

Monitoring bacterial growth Quantifying DNA or protein concentration Monitoring enzyme catalysis (kinetics) Detecting conformational changes in protein & nucleic acids Observing ligand-binding reactions

A student used western blotting to confirm the presence of horse Twist2 protein in osteoblast samples run on SDS-PAGE & transfered to a nitrocellulose membrane. The top image shows all interactions prior to the addition of substrate. The student finds the following reagents in the lab: Mouse anti-rabbit IgG-HRP Donkey anti-chicken IgG-AP Sheep anti-Mouse IgG-HRP TMB Which of the following antibodies did the student have the lab technician buy in order to successfully perform the western blot & what component of the image represents that antibody? 7

Mouse anti-Twist2; Red structure. Horse Twist2 protein is the antigen (black circle) The primary antibody (red structure) the student needs the lab tech to buy will need to be anti-Twist2. The available substrate is TMB. Therefore the 2ndary antibody (green Y with purple knobs) will have to be conjugated to HRP, giving 2 choices from the available reagents: Mouse anti-rabbit IgG-HRP Sheep anti-mouse IgG-HRP. To use these 2ndary antibodies, the primary antibody would have to come from a rabbit or a mouse. & the primary antibody has to be the red structure.

Effects of salt on spectral patterns and buffer components 9

Peaks and troughs can shift and/or change amplitude

The SDS-PAGE sample-loading buffer contains 62.5mM Tris pH 6.8, 4.5% SDS, 0.05% beta-mercaptoethanol, 50% glycerol, & 0.01% bromophenol blue. Match the function with each component. 6 ****************

SDS: disrupts 2ndary, tertiary, & quartenary structures of proteins & protein complexes Beta-mercaptoethanol: disrupts covalent bonds formed between 2 cysteine residues Glycerol: increases density of samples Bromophenol blue: monitors sample migration during electrophoresis

Effects of concentration on spectral patterns 9

Shape (peak/trough) essential remain unchanged

A student wants to use western blotting to confirm the presence of human Sonic hedgehog (Shh) in samples run on SDS-PAGE & transfered to a nitrocellulose membrane. The antibody reagent box in their lab contains Mouse anti-Shh MAb & Chicken anti-mouse IgG-HRP. What substrate will they need in order to perform colorimetric detection of Shh on their western blot? 7

TMB The enzyme linked to the 2ndary antibody is HRP, therefore TMB would be the substrate that could be converted to a colored product by that enzyme. (See table in lecture notes for further detail.)

If your protein of interest is positively charged in the transfer buffer, you will not be able to detect this protein with Western blot. 7

TRUE The protein of interest HAS to be (-) charged in the transfer buffer to migrate TOWARDS the membrane & then bind to the membrane. If your protein of interest is (+) charged, it will migrate away from the membrane during transfer.

Which of the following statements are true about both serial dilutions and step dilutions?

The stock solution is pipetted from only once in the series.

Why are plastic cuvettes not used at wavelengths below 250nm? 9

They interfere with the reading by absorbing light

Effects of handling cuvette incorrectly 9

Transmission Reflection (scratches on cuvette surface) Scattering (air bubbles or small partible-measures turbidity) Absorption (oil/fingerprints)

A highly positive charged protein will bind a cation exchanger and elute off by changing the pH.

True

Proteins are (-) charged & migrate towards the (+) electrode in SDS-PAGE. 6

True Negative proteins migrate towards the positive electrode. In an electric field, the cathode is negative (BLACK) & anode is positive (RED). Bigger proteins towards the top, smaller proteins towards the bottom. Migrates downwards towards the positive anode.

When handling, using & cleaning semi-micro cuvettes, which of the following will NOT have an effect on the sample's absorbance reading? 9

Turning the cuvette 180 degrees in the spectophotometer

Choosing a cuvette 9

UV quartz: 190-750nm (expensive) Methacrylate: ~260-750nm (inexpensive/disposable-interfere with DNA @260nm) Polystyrene: 350-750m (inexpensive/limited to Vis range) Glass: 340-750nm (inexpensive/limited to Vis range)

What are the typical spectophotometers? 9

UV-Vis 200-760nm Vis 400-760nm Infared >760

When handling, using & cleaning semi-micro cuvettes, which of the following will have an effect on the sample's absorbance reading? 9

Water droplets on the left window after cleaning Scratches on the light window caused while cleaning with a dry Kimwipe Air bubbles in the sample caused by pipetting too fast Fingerprints on the light window caused while handling

Common assay conditions that change enzyme pH and temp 9

[S] substrate concentration [E] enzyme concentration time course for reaction to occur *************

Traits of semi-micro cuvette 9

b= 1cm v= 350ul-3.5ul A semi micro cuvette volume holds samples of 0.35 mL - 1.7 mL (our lab uses semi-micro, 400uL of liquid for minimum volume for photometry.) To hold the smaller sample, these cuvettes must use a spacer, mount or standard cuvette exterior with a tapered interior. (For semi-micro)

Traits of standard cuvette 9

b= 1cm v>1,5ml ****What is B, and V? State standard path length of a cuvette as given. **** A semi micro cuvette volume holds samples of 0.35 mL - 1.7 mL (our lab uses semi-micro, 400uL of liquid for minimum volume for photometry.) A standard volume cuvette holds a measuring volume of 3.5 mL. To hold the smaller sample, these cuvettes must use a spacer, mount or standard cuvette exterior with a tapered interior. (For semi-micro)

As concentration increases, Absorbance 9

increases

Following an enzyme reaction via spectroscopy, look for _____ or ______ 9

loss of substrate or gain of product ***************************************

What are the typical sample size formats 9

microplate reader (single cell) spectrophotometer

When handling cuvettes, 1 should be careful not to touch. 9

the bottom side of the cuvette that the light passes thru

What happens to the A250-320 nm wavelength scan of a protein if you double the concentration? 9

the entire curve will increase by 2 fold

An undergraduate student compared the data from their assay performed today to data obtained on a previous day by the lab's star graduate student. The undergrad reported 0.020 ΔA340/5 secs. The grad student reported 0.160 ΔA340/min. Whose sample had more LDH activity?

the undergraduate student